Hemoglobin (Hb), in particular hemoglobin A1c (hereinafter, also referred to as HbA1c) of a form of glycosylated hemoglobins reflects an average blood sugar level in past 1 to 2 months. Therefore, hemoglobin A1c is widely used in a screening test for diabetes mellitus and as a test item for checking whether a diabetic keeps the blood sugar under control.
Conventionally, HbA1c has been measured by HPLC, immunoassay, electrophoresis or the like. Especially, HPLC is widely used in clinical examinations. HPLC requires only 1 to 2 minutes to measure each sample, and has achieved a measurement accuracy of about 1.0% in terms of a CV value obtained by a within-run reproducibility test. Measurement methods used for checking whether a diabetic keeps the blood sugar under control are required to perform at this level.
Meanwhile, application of an electrophoresis technique that enables high-accuracy measurement of HbA1c to the clinical examinations is expected to yield a significantly advantageous effect in cost because an electrophoresis apparatus has a simple configuration, and can be formed as a low-cost small system such as a microdevice electrophoresis system.
Measurement of Hb by electrophoresis has been used for a long time to separate abnormal Hbs with an unusual amino acid sequence. However, separation of HbA1c is significantly difficult, and takes 30 minutes or more by gel electrophoresis. Thus, electrophoresis has been unsatisfactory in terms of measurement accuracy and measurement time when applied to the clinical examinations. Therefore, electrophoresis has hardly been applied to clinical diagnosis of diabetes mellitus.
However, capillary electrophoresis, which was proposed in around 1990, generally enables high-accuracy measurement with high separation efficiency. For example, Patent Document 1 discloses a method for separating HbA1c by capillary electrophoresis.
However, use of the method of Patent Document 1 does not overcome the problem of taking a long time to measure, and also may denature Hb due to use of a buffer solution with a high pH of 9 to 12. For these reasons, it has been difficult to apply this method to the clinical examinations.
Patent Document 2 discloses a method using capillary electrophoresis in which an ionic polymer is allowed to flow through a capillary to dynamically coat the inner surface of the capillary with the ionic polymer, and a buffer solution containing a sulfated polysaccharide is used. This method enables measurement in a shorter time compared to gel electrophoresis, and takes only about 10 minutes to measure.
However, the coating layer formed by such a dynamic coating technique is significantly altered by each sample measurement, and this alteration prevents measurement of another sample without performing required procedure. In order to coat the inside of the capillary in the same way at the beginning of each measurement, it is necessary to remove the remaining coating layer by washing after each measurement, and to carry out the coating procedure again. Namely, for repetitive measurement, the washing and coating procedure needs to be performed between each measurement, resulting in an increase in the measurement time. The washing and coating procedure may cause a measurement error, and in addition requires a coating reagent to be prepared for the measurement, leading to a disadvantage in cost. Even when not used for the repetitive measurement, the technique takes about 10 minutes to measure, which is much longer than needed in HPLC, and is unsatisfactory for application to the clinical examinations.
For clinical diagnosis of diabetes mellitus, stable HbA1c, which is a type of HbA1c and used as a diabetic indicator, should be separated to remove effects of modified Hbs such as unstable HbA1c, carbamylated Hbs and acetylated Hbs. However, electropherograms obtained by the methods disclosed in Patent Document 1 and Patent Document 2 were unsatisfactory in terms of separation performance and measurement accuracy, and it has been difficult to separate stable HbA1c by the techniques within the scope of these methods.
Patent Document 1: Japanese Kohyo Publication No. Hei-09-510792(JP-T 09-510792)
Patent Document 2: Japanese Kokai Publication No. Hei-09-105739(JP-A 09-105739)